Fennel is a hardy, erect, umbelliferous herb of the family Apiaceae (Umbelliferae). Fennel has been used since ancient times as a flavouring agent in food. Essential oils and oleoresins derived from fennel are used in soaps, perfumes, creams, and liqueurs. Additionally, fennel has medicinal properties including uses as an antispasmotic, carminative, diuretic, expectorant, and laxative.
Fennel is an annual or perennial herb which can reach a height of 1.5 meters and has yellow flowers on a compound umbel. Two varieties of fennel are recognized that are thought to originate from subspecies capillaceum: Sweet or Roman fennel, subspecies capillaceum (Galib) Holmboe var. dulce Mill, and bitter or wild fennel, subspecies capillaceum (Galib) Holmboe var. vulgare Mill. Approximately 60% of the essential oil in the fennel plant is localized in the fruit, (commonly fennel seed) with the remaining portion lying within the other green parts of the plant. The oils of sweet and bitter fennel differ in their constitutive components. Bitter fennel oils are higher in fenchone or limonene and sweet fennel oils have a greater amount of anethone. The properties of sweet fennel oil are considered to be of higher quality due to a more pleasant aroma and flavour.
Foeniculum vulgare ssp. capillaceum is grouped into three varieties: Azoricum, also known as bulb fennel, Italian fennel, or Florence fennel, an annual that produces a bulb and is grown in Mediterranean countries where it is used as a vegetable. Dulce, also know as sweet fennel or French fennel, is mainly used as a condiment and Vulgare (bitter fennel), a perennial, has an essential oil content higher than that of dulce.
Fennel seed is used in the food industry as a flavouring agent for meats, vegetables, fish, soups, salad dressings, stews, breads, pastries, teas, and alcoholic beverages. The essential oils derived from fennel are used in condiments, soaps, creams, and perfume. The medicinal or nutraceutical applications of fennel include uses as an: antispasmodic, carminative, diuretic, expectorant, laxative, and stomachic. Additionally, fennel is used as a lactation stimulant, a remedy for colic, and as a treatment of gastroenteritis, hernia, indigestion, abdominal pain, and dissipation of phlegm.
Another species of the Apiaceae family is Caraway, Carum carvi L., a biennial herb which is native to Europe and Western Asia, but also grown on the prairie provinces of Canada. First year caraway plants resemble carrots, growing to about 8 inches tall with finely divided leaves and long taproots. By the second year, two to three foot stalks develop topped by umbels of white or pink flowers. The seeds are typically relatively small, brown and crescent shaped.
The Caraway seed is used whole as a spice or crushed to produce caraway oil. The seeds have a licorice flavour and are used in breads, soups, spreads, salad dressings, liqueurs, and the like. The leaves can be used in cooking, as can the roots. Caraway seeds and oil have medicinal applications for disorders such as rheumatism, eye infections, toothaches, and nausea. Caraway oil has some anti-bacterial properties.
The main constituent of caraway seed oil is carvone and limonene. Carvone has been used as a spice in foods, a sprouting inhibitor for potatoes and as a growth inhibitor for fungi and insects. The oil can also be used a fragrance component in cosmetics (e.g., soaps, creams, lotions, and perfumes).
Other species of the Apiaceae family include root crops (e.g., carrot, parsnip); stem, leaf, and petiole crops (e.g., celery, parsley); and seed crops (e.g., dill, anise, caraway). These species are used for foods, flavouring of foods, perfumes, medicines, and animal feed. Carrot is a major food crop, but can also be used as a food colouring agent in butter and as a sweetener of liqueurs. Anise is a licorice flavoured herb and is used to flavour liqueurs, candies, and toothpaste.
The use of doubled-haploid plants as a vehicle for plant breeding is well established and has become a routine practice for breeders of crops such as canola, wheat, barley, and maize. The main advantage of generating doubled-haploid plants from a cell culture is the greatly reduced time required to achieve homozygosity; years of selfing and recurrent selection are replaced by a single culture cycle. The use of haploid technologies results in the fixation of traits, allowing for efficient screening and selection of desirable phenotypes.
Haploid plants that comprise only a single set of chromosomes are infertile and must be doubled in their chromosome complement before use in breeding. Techniques for doubling the chromosome number in haploid plants using colchicine and other chemicals that disturb the cytoskeleton of cells are well known in the literature (e.g., Zhao et al., 1996).
There are several methods for generating doubled haploid plants. Haploid plants naturally occur with low frequency and can be identified in field grown populations based on examination of flower morphology. The low frequency of occurrence makes this approach impractical (See. e.g., U.S. Pat. No. 5,639,951). Haploid plants may also result from wide hybridization followed by chromosome elimination.
Wide hybridization was used to create Hordeum bulbosum by crossing common barley, Hordeum vulgare with H. bulbosum and the subsequent elimination of H. bulbosum chromosomes. Wide hybridization has been used to develop barley, wheat, maize, sorghum, and millet cultivars but has limited use outside of these cereal crops.
Another method for generating doubled haploid plants is gynogenesis. Gynogenesis involves the culture of female cells such as unfertilized ovaries or ovules. This method has only been shown to work with a few species and the frequency of embryo formation is low (See, e.g., U.S. Pat. No. 5,492,827).
Doubled-haploid plants can also be generated by androgenesis. Androgenesis involves culturing developing microspores with the entire anther or physically disrupting the anther and culturing the isolated microspores.
The development of embryos, haploid, and doubled-haploid plants from developing microspore in culture has been achieved to date in a variety of species representing many different genera (Dunwell, 1986; Ferrie et al., 1994). It is well known that a large variety of factors influence the success of inducing embryo development from isolated microspores or from anther cultures. (Ferrie and Keller, 1995; Maheshwari, et al., 1982).
One critical aspect of the methods for inducing embryo formation from microspores is to disrupt and shift the microspore developmental process using physical or chemical means. The disruption and shift must coincide with the developmental stage of the microspore that subsequently allows embryo formation. Typically the stage that is disrupted is the late uninucleate to early bi-nucleate stage of development (Gaillard et al., 1991; Kott et al., 1988; Fan et al., 1988). Historically, the chief agent for disruption was elevated temperatures, (Keller et al., 1978; Cordewener et al., 1994) but chemicals such as colchicine, cytochalasin B, and trifluralin that are known to disturb cellular cytoskeleton organization have more recently been shown to be effective as well (See e.g., U.S. Pat. Nos. 5,900,375; 6,200,808).
The nutrient medium is another aspect that has been shown to be important for recovery of embryos from induced microspores. Both the mineral composition of the medium and the percent of carbohydrates have been shown to be critical factors for some applications. High concentrations of sucrose (e.g., 13%) or other specific sugars such as maltose have been shown to be important. However, the optimal composition of the medium for embryo induction differs greatly from species to species. In addition to sugars and salts, plant growth regulators such as auxins, cytokinins and/or gibberellins may be required. Various gametocidal chemicals such as 2-hydroxynicotinic acid, 2-chloroethyl-phosphonic acid, and pronamide as well as undefined natural factors emanating from ovules (See e.g., U.S. Pat. Nos. 6,764,854; 6,362,393) may also be required components of the optimal nutrient medium.
There are vast differences between optimal nutrient media for the induction of embryos. In U.S. Pat. No. 4,840,906, spikes containing anthers were pretreated at 4° C. for a period of up to 28 days prior to culture of the barley microspores on media with varying sugar composition. This revealed the stimulative effect of maltose on the barley microspores. In U.S. Pat. Nos. 5,322,789 and 5,445,961, where isolated microspore and anther cultures of corn involved pre-treatment of microspores at 10° C., the requirement for mannitol and the chromosome doubling agent colchicine in the culture medium was demonstrated. These and other methods developed for cereal crops have the limitation that the methods may result in formation of significant numbers of albino plants.
U.S. Pat. No. 6,362,393 discloses a method for the production of doubled-haploid plants from wheat involved subjecting developing microspores to temperature and nutrient stress. A medium comprised of mannitol, maltose, auxins, cytokinins and/or gibberellin plant growth regulators, as well as a specific sporophytic development inducing chemical, were required for optimal embryo development. U.S. Pat. No. 6,764,854 describes an application of the above method for the production of doubled-haploid rice. U.S. Pat. No. 6,812,028 demonstrates a method for regeneration of isolated barley microspores that includes low temperature pretreatment, arabinogalactan protein, auxins and unknown natural factors from ovaries.
Brassinosteroids (BRs) are a group of plant growth-promoting substances that are similar to animal steroid hormones. They were first isolated from Brassica napus pollen in 1979 (Grove et al., 1979), but are known to be present in many plant species ranging from algae to higher plants. BRs are active at very low concentrations and can influence many plant growth and developmental processes, including cell elongation, cell division, and cell differentiation (Brosa, 1999). In addition to their role in plant growth and development, BRs have also been shown to protect plants from both abiotic and biotic stresses (Krishna, 2003). There are over 60 different BRs identified, with brassinolide (BL) and 24-epibrassinolide (EBR) being the most active of the known compounds for exogenous applications. These compounds have been used in plant tissue culture applications, leading to increases in the freezing and thermotolerance of cell suspensions (Wilen et al., 1995), induction of somatic embryogenesis in conifers and rice (Pullman et al., 2003), stimulation of shoot regeneration in B. oleracea var. botrytis and Spartina patens (Sasaki 2002; Lu et al., 2003), promotion of cell division in Chinese cabbage protoplasts (Nakajima et al., 1996), and increase in the rate of cell division in leaf protoplasts of Petunia hybrida (Oh et al. 1998). BRs have been tested in microspore embryogenesis of Brassica species and results showed an increase in embryogenesis (Ferric et al., 2005).
Isolated microspore culture protocols have been described for various Brassica species, (Ferrie et al., 1995, 1999, 2004; Barro et al. 1999; and Lionneton 2001). Factors that have been identified that contribute to induction and development of microspore-derived embryos included growth conditions of the parent plants, stage of microspore development, temperature stress, osmotic stress, and carbohydrate composition of the medium. The requirement for temperature stress may be replaced by chemical inhibitors of cytoskeleton integrity (See, e.g., U.S. Pat. Nos. 5,900,375 and 6,200,808).
Despite the successful development of embryos from microspores of numerous species, many species remain unresponsive. Arabidopsis thaliana is an example of a recalcitrant species that does not respond to methods that are known to succeed for the closely related Brassica species. Additionally, it is well known by those of ordinary skill in the art that response to microspore culture varies from cultivar to cultivar and from plant to plant of the same cultivar, suggesting unknown genetic influences.